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Behavioral and neurochemical effects of dietary methyl donor deficiency

combined with unpredictable chronic mild stress in rats

Article  in  Behavioural Brain Research · December 2013

DOI: 10.1016/j.bbr.2013.11.047 · Source: PubMed

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 Behavioural Brain Research 261 (2014) 8–16

Contents lists available at ScienceDirect

Behavioural Brain Research

journal homepage: www.elsevier.com/locate/bbr

Research report

Behavioral and neurochemical effects of dietary methyl donor

deficiency combined with unpredictable chronic mild stress in rats
H. Javelot a,b,c,∗ , M. Messaoudi b , C. Jacquelin a , J.F. Bisson b , P. Rozan b , A. Nejdi b ,
C. Lazarus d , J.C. Cassel d , C. Strazielle a,e , R. Lalonde f
a
Laboratoire de Nutrition Génétique et Exposition aux Risques Environnementaux, INSERM U954, Faculté de Médecine de Nancy – UHP,
Vandœuvre-lès-Nancy, France
b
ETAP-Applied Ethology – Neuropsychopharmacology Department, Vandœuvre-lès-Nancy, France
c
Clinical Pharmacy Service – Mental Health Establishment (EPSAN), Brumath, France
d
Laboratoire de Neurosciences Cognitives et Adaptatives, UMR 7363, Université de Strasbourg, CNRS, Strasbourg, France
e
Laboratoire de Microscopie Electronique, Faculté de Médecine de Nancy – UHP, Vandœuvre-lès-Nancy, France
f
Université de Rouen, Dépt. Psychologie, Laboratoire ICONES (EA 4699), Mont-Saint-Aignan, France

h i g h l i g h t s

• Methyl donor deficient diet caused disinhibiton.

• Chronic stress exacerbated methyl donor deficiency effects on locomotors activity.
• Hyperhomocysteinemia may contribute to monoaminergic and behavioral disturbances.

a r t i c l e i n f o a b s t r a c t

Article history: Methyl donor deficiencies and chronic stress cause depression independently, but their interaction has
Received 15 May 2013 never been thoroughly evaluated. In our study, methyl donor deficient diet and chronic stress condition
Received in revised form consisted respectively of a B2, B9, B12, and choline-free diet and a chronic mild stress procedure. Rats were
22 November 2013
randomly assigned to six groups with three “diet” conditions (free-feeding, pair-fed and methyl donor
Accepted 26 November 2013
Available online 9 December 2013
deficient diet) and two “stress” conditions (no-stress and stress) and were evaluated in the open-field,
the elevated plus-maze and the forced swimming test. After the behavioral evaluation, corticosterone
and homocysteine plasma levels were measured and dopamine, DOPAC, serotonin, 5HIAA concentra-
Keywords:
Rat tions were evaluated in several brain areas. Rats given a methyl donor deficient diet for 11 weeks causing
Behavior elevated plasma homocysteine levels were compared to pair-fed and free-feeding rats with or without
Depression unpredictable chronic mild stress. Regardless of stress environmental conditions, the methyl donor defi-
Chronic mild stress cient diet decreased plasma corticosterone levels and caused disinhibition in the elevated plus-maze
B-vitamin condition relative to both control groups. However, stress potentiated the effects of the deficient regi-
Methyl donors men on rearing in the open-field and climbing in the forced swim test. The dietary changes involved in
behavior and plasma corticosterone could be caused by homocysteine-induced decreases in dopamine
and 5-hydroxytryptamine metabolites in selective brain regions and it can be noted that regardless of
stress-conditions, methyl donor deficient diet decreases DOPAC/dopamine and 5HIAA/serotonin ratios in
striatum and hypothalamus and selectively 5HIAA/serotonin ratio in the sensorimotor cortex. Our exper-
imental data is particularly relevant in the context of neuropsychiatric disorders frequently associated
with folate deficiency and hyperhomocysteinemia.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The neuropsychiatric consequences of folate and vitamin B12

deficiencies have been extensively demonstrated [1,2]. Despite
some degree of overlap, folate deficiency has been more fre-
quently associated with depression and cobalamin deficiency
∗ Corresponding author at: Clinical Pharmacy Service – Mental Health Establish-
with psychosis [3]. More generally, methyl donor deficiencies
ment (EPSAN), Brumath, France. Tel.: +33 0388646170; fax: +33 0388646158.
E-mail address: herve.javelot@ch-epsan.fr (H. Javelot).
lead to several psychiatric disorders [4]. Genetic data shows

0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbr.2013.11.047
 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16
a close link between folate metabolism and depression [4,5].
According to a meta-analysis, the common C677T polymorphism
of methylenetetrahydrofolatereductase (MTHFR), catalyzing the
conversion of 5,10-methylenetetrahydrofolate to the bio-active 5-
methyltetrahydrofolate (5-MTHF), decreased 5-MTHF production,
decreased serum folate levels, and increased the risk of depression
[6].
Today, a large number of converging data suggests that
there is a relationship between folate deficiency and depression,
and hyperhomocysteinemia might be its metabolic consequence
[4,5]. Two major metabolites could be involved in the neuro-
chemical hypothesis of folate-related depression: (1) 5-MTHF,
which influences tetrahydrobiopterin (BH4) levels and activity
and (2) S-adenosylmethionine (SAMe), a methyl donor in numer-
ous biochemical reactions, both consistent with the monoamine
hypothesis of depression, since BH4 is an essential cofactor for
5-hydroxytryptamine (5HT), dopamine, and noradrenaline syn-
thesis and methylation is a limiting step in the latter [4,5].
Hyperhomocysteinemia could counteract optimal biosynthesis of
neurotransmitters via oxidative stress [4,7]. 5-MTHF and SAMe
possess antidepressant-like activity [5,7,8]. Low serum folate
levels may be involved in resistant-depression episodes which
present with poor responses to fluoxetine, whereas folates and
their derivatives have antidepressant properties even when there
is no measurable folate deficiency, highlighting their action as
monoamine enhancers [4,9,10].
Few studies have evaluated the effect of folate derivatives
or SAMe on mood-related behaviors in rodent models [11–18],
and even fewer data is available on the effects of methyl donor
deficient diets on mood [19]. Brocardo et al. [16] demonstrated
antidepressant-like effects of folate in the forced swim test in
mice, which seemed to involve glutamatergic n-methyl-d-asparate
(NMDA) receptors, the l-arginine-nitric oxide-cyclic guanosine
monophosphate pathway, or opioids [17,18]. Benelli et al. [12]
showed that SAMe had a better antidepressant-like action than
imipramine in the unpredictable chronic mild stress (UCMS), a
well-documented model of depression [20]. Moreover, Genedani
et al. [13] showed that the antidepressant action of SAMe was
partially related to its ability to restore polyamine levels follow-
ing UCMS in nucleus accumbens and hippocampus. Lalonde et al.
[19] described hyperactivity without change in anxiety levels,
motor coordination, and spatial learning after five-month of dietary
restriction of the methyl donors vitamins B2, folate, B12, and
choline. In the context of depressive subtypes, Fraguas et al. [21]
showed that in major depressive disorder with aggressive behavior,
homocysteine levels were positively correlated with the severity
and the duration of the depressive episode, and this was not found
in major depression without aggressive symptoms. Mineur et al.
[21] consider the UCMS model as being particularly relevant in
depressed patients presenting with aggressive behavior.
In the present study, we assessed the separate and combined
effects of UCMS with the same methyl donor deficient diet used
previously in mice [19], lacking vitamin B2, folate, vitamin B12,
and choline, similar to the one used to analyze rat exploratory
activity in the elevated plus-maze and other tests [23]. In addition
to the elevated plus-maze [24], rats explored the open-field [25]
and were then exposed to the forced swim test of depression-like
behavior [26]. We hypothesized that mild stress over several weeks
would potentiate the effects of the methyl donor deficient diet,
possibly increasing anxiety and depression-like behaviors. At the
end of the behavioral evaluation, corticosterone and homocysteine
plasma levels were measured along with dopamine and 5HT con-
centrations in several brain areas. We hypothesized that mild stress
would also potentiate the effects of a methyl donor deficient diet
at the biochemical level, possibly increasing plasma corticosterone
and homocysteine levels as well as brain monoamine levels.
9
2. Materials and methods
2.1. Animals
One-month-old Wistar rats (Harlan, The Netherlands) were
housed in groups of four inside 48 cm × 27 cm × 20 cm poly-
carbonate cages (UAR, Epinay-sur-Orge, France) under controlled
conditions with respect to ambient humidity (50 ± 5%) and temper-
ature (22 ± 1 ◦ C). The rats were maintained on a 12 h light/12 h dark
cycle (on at 20.00 pm-08.00 am). After a 7-day adaptation period,
rats were randomly assigned to one of three diet groups with or
without UCMS (n = 8). The deficient diet lacking vitamins B2, B9,
B12, and choline (Special Diet Services, Saint-Gratien, France) was
given for eight weeks without behavioral testing and then for three
more weeks with behavioral testing and compared with appro-
priate pair-fed and free-feeding controls. At the same time each
week, the rats were weighed and their food and water intake mon-
itored, the final measure taken on day 77. The deficient animals
were compared with two pair-fed groups receiving a standard diet
(M20, Special Diet Services). Food eaten by a vitamin-B-deprived rat
was weighed each day at the same hour and the means obtained
were then used for feeding the pair-fed rats. The free-feeding
group ate the same diet as the pair-fed animals but ad libitum.
The experiment follow the guidelines provided by the ASAB Eth-
ical Committee for the use of animals in research (Anim. Behav.
2006, 71, pp. 245–254) and the Canadian Council on Animal Care
(Guide to the Care and Use of Experimental Animals), 2nd ed., vol. 2
(1993); 1st ed., vol. 1 (1984). All procedures were also in compliance
with regulations provided by the European Community Council
Directive of 24 November 1986 (86/609/EEC).
2.2. Unpredictable chronic mild stress
The three non-stressed groups remained in the same room,
while the three stressed groups were moved from Monday to Fri-
day to a separate one and exposed to four different stressors in a
different order each week: intermittent white noise (85 dB; 10.0
apm–16.00 pm), cage tilt (10.0 apm–16.00 pm), continuous light-
ing (24 h), and exposure to soiled bedding inside the cage (200 ml
of spilled water).
2.3. Behavioral methods and procedures
2.3.1. Open-field
The rats were first tested after 8 weeks of diet exposure (day
56) in the open-field. Each rat was individually placed for 3 min in
the center of a circular arena, 60 cm in diameter, with clear plex-
iglas walls at a height of 40 cm and a plexiglas floor divided into
9 equal areas: 8 black areas at the periphery and a white one at
the center. The number of squares crossed and the number of rears
were recorded under dim white light, as well as the time spent in
the central square (s). The floor was washed with a water–alcohol
solution and dried before each subject to diffuse odor cues.
2.3.2. Elevated plus-maze
The elevated plus-maze was made of black plasticized wood,
consisting of four cross-shaped arms, two open (50 cm × 12 cm) and
two enclosed on opposite sides, with the walls of the enclosed arms
reaching 40 cm in height, together with a common central platform
(12 cm × 12 cm). On day 58, each rat was placed in the center of the
maze with its head facing an open arm and left to explore it in
a single 5-min session. The test was conducted under dim white
light and behaviors recorded with a CCD-TV camera. The numbers
of entries (4-paw criterion) inside open and enclosed arms were
10 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16
measured together with the time spent inside each arm and the
open/total arm duration ratio.
2.3.3. Forced swim test
During the pre-test session on day 75, the rats were placed inside
a plexiglas cylinder (50 cm height and 20 cm diameter) filled with
water at a temperature of 25 ◦ C to a depth of 30 cm for 15 min.
The water was changed on every trial to eliminate urine, feces, and
other possible odor clues left by the previous rat. After the swim
session, the rats were removed from the cylinder, gently dried with
towels, and placed under a red light (30 ◦ C) for 20 min before being
returned to their home cage. The following day, the rats were sub-
mitted to the same swim stress condition for 5 min. The 5-min test
session was recorded with a CCD-TV camera, and the duration of
immobility, swimming, and climbing behaviors were scored later
on. Immobility was defined as the absence of any movement, with
the body inclined forward, passively floating, with immobile paws.
Swimming was defined as horizontal movements in the water, and
climbing as vertical movements similar to escape attempts.
2.4. Plasma concentrations of homocysteine and corticosterone
On day 77, the animals were euthanized with halothane. Blood
was rapidly removed by cardiac puncture and sampled into tubes
with EDTA, then centrifuged (4 ◦ C, 2500 ␮g, 15 min) to retrieve
the plasma fraction. To reduce the effects of diurnal variations on
titration, the rats in each group were killed alternatively within a
4 h time-frame and blood samples were collected between 14:00
PM and 18:00 PM. Plasma was stored at −80 ◦ C until homocys-
teine and corticosterone concentrations were respectively assessed
by fluorescent polarization immunoassay (IMX system; Abbott,
Fornebu, Norway, [27]) and radioimmunoassay. Plasma was first
deproteinized by ethanol extraction. Regarding corticosterone,
the working range of the assay was 20–10,000 pg/tube and con-
centrations were estimated from the parameters of a standard
curve linearized by logit-log transformation [28]. Corticosterone
and homocysteinemia were respectively expressed in mg/ml and
ng/ml.
2.5. Brain and spinal concentrations of biogenic amines and
metabolites
Immediately after cardiac puncture, the rats were decapi-
tated and their brains removed. Brain tissue samples were put
in nitrogen liquid before being weighed and kept at a −80 ◦ C
temperature; hippocampus, limbic cortex, sensorimotor cortex,
striatum, thalamus, hypothalamus, cerebellum, and medulla were
then analyzed at a neurochemical level. Left and right struc-
tures from each rat were pooled for bilateral brain regions. The
concentrations of dopamine and one of its major metabolites,
3,4-dihydroxyphenylacetic acid (DOPAC), along with 5HT and its
principal metabolite, 5-hydroxyindolacetic acid (5-HIAA), were
measured by means of high performance liquid chromatography
(HPLC) with electrochemical detection. Tissue samples were pre-
pared for HPLC by homogenization in 1 N formic acid/acetone
(18:8.5, v/v), and the formic extracts used for monoamine deter-
minations without further purification. The HPLC system consisted
of an ESA liquid chromatography pump (ESA Inc., Bedford) coupled
to an ESA Coulochem II detector (Eurosep Instruments) equipped
with a 5014 high performance analytic cell (ESA Inc., Bedford) set
at +0.4 V and analyzed on a C18 Spherisorb ODS2 reverse phase
column (5 ␮m pore size, 4.6 mm in diameter, 25 cm in length).
The mobile phase consisted of 0.1 M NaH2 PO4 , pH = 3, containing
0.1 mM/l of EDTA, 1.7 mM/l 1-octane sulfonic acid sodium salt, and
10% acetonitrile. The flow rate was 1 ml/min. Concentrations of the
different compounds were determined with data analysis software
(Baseline 810, Waters) and expressed in ng/mg microwaved tissue.
2.6. Statistical analyses
For non-repeated measures, 3 × 2 analyses of variance
(ANOVA)s were conducted with diet at 3 levels and stress at
2 levels as the main factors. For repeated measures, three-way
ANOVAs with repeated measures on the final factor were con-
ducted, with diet as one main factor, stress as the second main
factor, and time as the repeated measure. Whenever the interac-
tion was significant, Fisher’s least significant difference (LSD) test
was used to determine between-group differences.
3. Results
3.1. Exploratory activity and forced swimming
3.1.1. Open-field (see Table 1)
Stress exerted an effect on rearing in the open-field
[F(1,42) = 4.08; P = 0.05] and interacted with diet for this measure
as well as time spent inside the central square [F(2,42) = 4.24;
P = 0.02; F(2,42) = 5.19; P = 0.01, respectively]. Among stressed rats,
the methyl donor deficient group reared more often than either
pair-fed or free-feeding groups [P < 0.05 and P < 0.01, respectively,
Fisher’s test]. This result was not found among non-stressed rats
[P > 0.05]. Regarding the time spent inside the central square, the
stressed pair-fed group had higher values than the other two
stressed groups [P < 0.05], whereas no difference was found among
the non-stressed.
3.1.2. Elevated plus-maze (see Table 1)
In the elevated plus-maze, there were significant diet effects
on time spent inside open arms [F(2,35) = 5.06; P = 0.01] and
enclosed arms [F(2,35) = 4.82, P = 0.01] as well as open arm
entries [F(2,35) = 6.76, P = 0.003] and open/total arm duration
[F(2,35) = 5.06; P = 0.01]. Specific comparisons revealed that methyl
donor deficient rats entered more often and spent more time inside
open arms and had higher open/total arm durations than either
pair-fed or free-feeding rats [P < 0.05, Fisher’s test] and conversely
spent less time inside enclosed arms [P < 0.05]. Methyl donor defi-
cient rats also tended to spend more time inside the central square
than these groups, but the comparison with the free-feeding group
did not quite reach the required level of significance [P = 0.06].
Stress had no impact on any of these measures (Table 2).
3.1.3. Forced swim test (see Fig. 1)
During the test period, diet effects were found on swimming and
climbing duration as well as climbing frequency [F(2,42) = 22.56,
P < 0.0001; F(2,42) = 17.05, P < 0.0001; F(2,42) = 10.4, P = 0.0002,
respectively]. There were also diet × stress interactions for all three
measures [F(2,42) = 10.41, P = 0.0002; F(2,42) = 8.33, P = 0.0009;
F(2,42) = 10.75, P = 0.0002, respectively]. Among stressed rats, the
methyl donor deficient group spent less time swimming, more
time climbing, and climbed more often than the other two groups
[P < 0.0001, Fisher’s test]. No such result was found among non-
stressed rats and no effect was found for immobility time.
3.2. Plasma homocysteine and corticosterone levels (see Fig. 2A
and B)
3.2.1. Homocysteine
There were diet, stress, and interaction effects between
these factors on homocysteine levels [F(2,30) = 70.71, P < 0.0001;
F(1,30) = 4.94, P = 0.03 and F(2,30) = 5.59, P = 0.009, respectively].
 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16 11

Table 1
Effects of UCMS and methyl donor deficiency on exploratory activity in the open-field and plus-maze.

Free-feeding Free-feeding Pair-fed Pair-fed Methyl donor Methyl donor

Non-stress Stress Non-stress Stress deficient deficient
Non-stress Stress

Open-field
Squares crossed 51.9 ± 3.6 44.1 ± 3.6 51.4 ± 1.6 56 ± 4.4 50.5 ± 2.2 58.1 ± 4.4
Rears 8 ± 1.8 6.2 ± 6.9 6.1 ± 1 8.1 ± 1.6 6.6 ± 1.6 15.9 ± 2.6* , ##
Central square duration (s) 3.4 ± 0.8 2.7 ± 1 2.4 ± 0.6 8.5 ± 2.5 4.4 ± 1.3 2.5 ± 1.8*

Elevated plus-maze
Central square visits 13.4 ± 2.5 13.5 ± 2.4 13.7 ± 0.9 20.2 ± 1.5 12.9 ± 1.5 15.5 ± 1.7
Central square duration (s) 68.8 ± 16.4 66.5 ± 10.7 61.9 ± 4.7 70 ± 11.2 72.25 ± 11.5 100.6 ± 4.7
Open arm visits 0.8 ± 0.4 0.7 ± 0.3 1.2 ± 0.4 0.7 ± 0.5 2.4 ± 0.6† , ‡ 2.7 ± 0.7† , ‡
Open arm duration (s) 5.8 ± 3.3 8.1 ± 3.9 8.2 ± 3.7 6.2 ± 3.7 21.6 ± 5.5†† , ‡‡ 22.4 ± 8†† , ‡‡
Enclosed arm duration (s) 225.4 ± 17.8 225.4 ± 13.7 229.9 ± 7.9 223.7 ± 12.7 206.1 ± 15.2† , ‡ 177 ± 9.8† , ‡
Open/total arm duration 2.68 ± 1.61 3.96 ± 1.96 3.65 ± 1.66 2.82 ± 1.77 10.13 ± 2.62† , ‡ 11.34 ± 11.76† , ‡

All data were expressed as the mean ± SEM.

*
P < 0.05 methyl donor deficient vs pair-fed in stressed rats.
##
P < 0.01 vs free-feeding.
†
P < 0.05.
††
P < 0.01 methyl donor deficient vs pair-fed.
‡
P < 0.05.
‡‡
P < 0.01 vs free-feeding (stressed and non-stressed rats pooled); all comparisons with Fisher’s test.

Fig. 1. Effects of UCMS and methyl donor deficiency on the duration of immobility (A) and swimming (B) and the duration and frequency of climbing (C and D, respectively)
in the forced swimming test during the test session. Results are expressed in mean ± SEM. ***P < 0.001 and *P < 0.05 methyl donor deficient vs pair-fed; ### P < 0.001 and
#
P < 0.05 vs free-feeding, in stressed groups (Fisher’s test).
12 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16

Methyl donor deficient rats expressed a higher level of homo-

cysteinemia than pair-fed and free-feeding rats independently of
stress levels [P < 0.0001, Fisher’s test], though the effect was ampli-
fied in non-stressed rats.

3.2.2. Corticosterone
There were diet [F(2,30) = 7.84, P = 0.002] and stress
[F(1,30) = 6.04, P = 0.02] effects on corticosterone levels. Methyl
donor deficient rats expressed a lower level of corticosterone
levels than pair-fed and free-feeding rats [P < 0.01 and P < 0.05,
respectively, Fisher’s test], while stress increased these levels.

3.3. Biogenic amines and metabolites in brain (see Table 3)

3.3.1. DOPAC
Two-way ANOVAs revealed diet effects on DOPAC levels in the
thalamus, the hypothalamus, and the cerebellum [F(2,42) = 5.21,
P = 0.009; F(2,41) = 3.41, P = 0.04; F(2,42) = 3.66, P = 0.03, respec-
tively] as well as stress effects on the medulla [F(1,41) = 5.34,
P = 0.03]. Methyl donor deficient rats pooled for the stress variable
had lower DOPAC levels than the other groups of rats in hypothala-
mus [P < 0.05], but in thalamus and cerebellum their levels were
only lower than free-feeding rats [P < 0.01 and P < 0.05, respec-
tively]. The stress effect on the medulla is due to higher DOPAC
levels in UCMS-exposed rats.
Fig. 2. Effects of methyl donor deficiency on plasma corticosterone (ng/ml)
and homocysteine levels (␮mol/l). (A) Corticosterone levels (ng/ml). Results are
3.3.2. Dopamine expressed in mean ± SEM. †† P < 0.01 methyl donor deficient vs pair-fed; ‡ P < 0.05 vs
free-feeding (non-stressed and stressed rats pooled; comparisons with Fisher’s test).
There were diet effects on dopamine levels in the medulla
(B) Homocysteine levels (␮mol/l). Results are expressed in mean ± SEM. ***P < 0.001
[F(2,41) = 4.22, P = 0.02] and a significant diet × stress interaction methyl donor deficient vs pair-fed; #### P < 0.05 vs free-feeding, in stressed groups
in the sensorimotor cortex [F(2,42) = 4.97, P = 0.01]. Methyl donor (Fisher’s test).
deficient rats expressed higher dopamine levels in medulla only
with respect to pair-fed rats [P < 0.05]. In the sensorimotor cortex,

Table 2
Effects of UCMS and methyl donor deficiency on dopamine and DOPAC levels (in ng/mg) and the ratio DOPAC/dopamine in different brain regions and cervical spinal cord.

Free-feeding Free-feeding Pair-fed Pair-fed Methyl donor Methyl donor

Non-stress Stress Non-stress Stress deficient deficient
Non-stress Stress

DA
Hippocampus 0.128 ± 0.039 0.069 ± 0.058 0.064 ± 0.036 0.105 ± 0.036 0.099 ± 0.027 0.136 ± 0.053
Limbic cortex 0.075 ± 0.004 0.065 ± 0.006 0.062 ± 0.003 0.059 ± 0.004 0.060 ± 0.009 0.064 ± 0.007
Sensorimotor cortex 0.341 ± 0.048 0.290 ± 0.027 0.189 ± 0.062 0.377 ± 0.043 0.316 ± 0.045 0.225 ± 0.054
Striatum 1.141 ± 0.067 1.135 ± 0.044 1.169 ± 0.043 1.054 ± 0.138 1.175 ± 0.081 1.314 ± 0.121
Thalamus 0.114 ± 0.032 0.095 ± 0.012 0.058 ± 0.005 0.118 ± 0.035 0.086 ± 0.015 0.068 ± 0.011
Hypothalamus 0.510 ± 0.101 0.595 ± 0.101 0.642 ± 0.144 0.696 ± 0.157 0.546 ± 0.113 0.534 ± 0.085
Cerebellum 0.008 ± 0.001 0.008 ± 0.001 0.007 0.006 0.006 0.008
Medulla 0.057 ± 0.004 0.061 ± 0.004 0.050 ± 0.003 0.062 ± 0.005 0.064 ± 0.004† 0.068 ± 0.004†,

DOPAC
Hippocampus 0.034 ± 0.008 0.022 ± 0.006 0.024 ± 0.006 0.034 ± 0.006 0.025 ± 0.004 0.027 ± 0.003
Limbic cortex 0.038 ± 0.005 0.037 ± 0.006 0.031 ± 0.002 0.030 ± 0.002 0.029 ± 0.003 0.035 ± 0.003
Sensorimotor cortex 0.081 ± 0.019 0.064 ± 0.009 0.049 ± 0.015 0.106 ± 0.022 0.078 ± 0.013 0.057 ± 0.020
Striatum 0.554 ± 0.044 0.572 ± 0.024 0.589 ± 0.021 0.466 ± 0.075 0.503 ± 0.047 0.455 ± 0.027
Thalamus 0.042 ± 0.04 0.043 ± 0.004 0.058 ± 0.005 0.042 ± 0.004 0.035 ± 0.003‡‡ 0.029 ± 0.003‡‡
Hypothalamus 0.127 ± 0.016 0.159 ± 0.022 0.152 ± 0.027 0.149 ± 0.024 0.096 ± 0.016†,‡ 0.104 ± 0.016†,‡
Cerebellum 0.013 ± 0.001 0.015 ± 0.002 0.014 ± 0.002 0.012 ± 0.001 0.010 ± 0.001‡ 0.012 ± 0.001‡
Medulla 0.015 ± 0.001 0.016 ± 0.001 0.012 ± 0.001 0.017 ± 0.002 0.015 ± 0.001 0.016 ± 0.001

DOPAC/DA
Hippocampus 0.471 ± 0.144 0.535 ± 0.142 0.660 ± 0.136 0.515 ± 0.116 0.671 ± 0.287 0.607 ± 0.181
Limbic cortex 0.507 ± 0.059 0.581 ± 0.083 0.497 ± 0.033 0.535 ± 0.060 0.523 ± 0.049 0.577 ± 0.067
Sensorimotor cortex 0.225 ± 0.027 0.221 ± 0.034 0.299 ± 0.031 0.272 ± 0.025 0.262 ± 0.030 0.294 ± 0.067
Striatum 0.483 ± 0.020 0.505 ± 0.014 0.507 ± 0.024 0.430 ± 0.042 0.426 ± 0.022††,‡‡‡ 0.360 ± 0.025††,‡‡‡
Thalamus 0.469 ± 0.067 0.473 ± 0.043 0.584 ± 0.041 0.502 ± 0.089 0.448 ± 0.043 0.459 ± 0.041
Hypothalamus 0.272 ± 0.031 0.278 ± 0.019 0.250 ± 0.014 0.232 ± 0.014 0.184 ± 0.010†,‡ 0.208 ± 0.022†,‡
Cerebellum 1.732 ± 0.111 2.004 ± 0.282 2.016 ± 0.266 1.879 ± 0.082 1.803 ± 0.163 1.436 ± 0.125
Medulla 0.269 ± 0.016 0.269 ± 0.016 0.244 ± 0.012 0.269 ± 0.017 0.245 ± 0.017 0.245 ± 0.007

All data were expressed as the mean ± SEM.

Specific comparisons with non-stressed and stressed rats pooled with Fisher’s test: † P < 0.05 and †† P < 0.01 methyl donor deficient vs pair-fed; ‡ P < 0.05, ‡‡ P < 0.01 and ‡‡‡ P < 0.001
vs free-feeding.
ANOVA:  P < 0.05 stressed rats vs non-stressed rats.
 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16 13

Table 3
Effects of CMS and methyl donors deficiency on 5-HT and 5HIAA levels (in ng/mg) and the ratio 5HIAA/5-HT in different brain regions and cervical spinal cord.

Free-feeding Free-feeding Pair-fed Pair-fed Methyl donor deficient Methyl donor deficient
Non-stress Stress Non-stress Stress Non-stress Stress

5HT
Hippocampus 0.204 ± 0.013 0.184 ± 0.015 0.246 ± 0.025 0.222 ± 0.016 0.204 ± 0.025 0.204 ± 0.011
Limbic cortex 0.452 ± 0.015 0.418 ± 0.016 0.448 ± 0.006 0.483 ± 0.021 0.442 ± 0.049 0.479 ± 0.036
Sensorimotor cortex 0.214 ± 0.016 0.160 ± 0.019 0.175 ± 0.018 0.221 ± 0.021 0.207 ± 0.012 0.204 ± 0.029
Striatum 0.344 ± 0.038 0.397 ± 0.024 0.417 ± 0.020 0.330 ± 0.036 0.418 ± 0.031 0.339 ± 0.016
Thalamus 0.417 ± 0.012 0.404 ± 0.021 0.378 ± 0.016 0.400 ± 0.019 0.385 ± 0.026 0.340 ± 0.028
Hypothalamus 0.764 ± 0.050 0.766 ± 0.031 0.779 ± 0.021 0.725 ± 0.132 0.750 ± 0.050 0.688 ± 0.053
Cerebellum 0.026 ± 0.003 0.031 ± 0.003 0.032 ± 0.003 0.020 ± 0.003 0.031 ± 0.002 0.035 ± 0.004
Medulla 0.353 ± 0.017 0.332 ± 0.010 0.336 ± 0.020 0.373 ± 0.021 0.403 ± 0.038††,‡‡‡ 0.407 ± 0.016††,‡‡‡

5HIAA
Hippocampus 0.184 ± 0.013 0.166 ± 0.012 0.218 ± 0.022 0.195 ± 0.014 0.190 ± 0.023 0.185 ± 0.006
Limbic cortex 0.219 ± 0.007 0.201 ± 0.011 0.216 ± 0.008 0.216 ± 0.010 0.197 ± 0.022 0.197 ± 0.011
Sensorimotor cortex 0.153 ± 0.010 0.128 ± 0.010 0.139 ± 0.012 0.139 ± 0.009 0.131 ± 0.008 0.122 ± 0.011
Striatum 0.263 ± 0.018 0.298 ± 0.012 0.342 ± 0.024 0.237 ± 0.025 0.282 ± 0.018 0.239 ± 0.010
Thalamus 0.322 ± 0.008 0.292 ± 0.014 0.326 ± 0.017 0.310 ± 0.013 0.288 ± 0.011††,‡ 0.250 ± 0.024††,‡
Hypothalamus 0.424 ± 0.024 0.423 ± 0.014 0.432 ± 0.029 0.405 ± 0.055 0.381 ± 0.036 0.350 ± 0.033
Cerebellum 0.032 ± 0.002 0.029 ± 0.002 0.030 ± 0.003 0.028 ± 0.001 0.027 ± 0.002 0.054 ± 0.024
Medulla 0.145 ± 0.007 0.150 ± 0.006 0.146 ± 0.008 0.156 ± 0.011 0.169 ± 0.005‡ 0.162 ± 0.005‡

5HIAA/5HT
Hippocampus 0.906 ± 0.039 0.917 ± 0.057 0.917 ± 0.084 0.883 ± 0.042 0.940 ± 0.054 0.922 ± 0.042
Limbic cortex 0.485 ± 0.010 0.482 ± 0.023 0.483 ± 0.016 0.449 ± 0.016 0.433 ± 0.029 0.417 ± 0.016
Sensorimotor cortex 0.723 ± 0.021 0.821 ± 0.036 0.803 ± 0.032 0.648 ± 0.037$$ 0.633 ± 0.022***,# 0.618 ± 0.022*,###
Striatum 0.798 ± 0.048 0.761 ± 0.042 0.820 ± 0.039 0.725 ± 0.023 0.680 ± 0.020†,‡ 0.708 ± 0.016†,‡
Thalamus 0.774 ± 0.014 0.730 ± 0.038 0.880 ± 0.074 0.776 ± 0.021 0.759 ± 0.029 0.739 ± 0.038
Hypothalamus 0.561 ± 0.026 0.556 ± 0.019 0.556 ± 0.036 0.585 ± 0.033 0.502 ± 0.021†,‡ 0.507 ± 0.025†,‡
Cerebellum 1.545 ± 0.406 0.973 ± 0.033 0.952 ± 0.071 1.826 ± 0.435 0.872 ± 0.066 1.719 ± 0.835
Medulla 0.412 ± 0.012 0.453 ± 0.012 0.434 ± 0.009 0.416 ± 0.012$$ 0.391 ± 0.016* 0.400 ± 0.005###

All data were expressed as the mean ± SEM.

Specific comparisons in non-stressed or stressed rats with Fisher’s test: * P < 0.05 and *** P < 0.001 methyl donor deficient vs pair-fed; # P < 0.05 and ### P < 0.001 vs free-feeding
and $$ P < 0.01 pair-fed vs free-feeding.
Specific comparisons with non-stressed and stressed rats pooled with Fisher’s test: † P < 0.05 and †† P < 0.01 methyl donor deficient vs pair-fed; ‡ P < 0.05 and ‡‡‡ P < 0.001 vs
free-feeding.

methyl donor deficient rats had lower dopamine levels than
pair-fed rats among stressed groups and pair-fed rats had lower
levels than free-feeding rats among non-stressed groups.
3.3.3. DOPAC/dopamine ratio
There were diet effects on the DOPAC/dopamine ratio in the
striatum and the hypothalamus [F(2,42) = 8.39, P = 0.0009; F(2,41)
=8, P = 0.001, respectively]. Methyl donor deficient rats had a lower
ratio than either pair-fed or free-feeding rats in the striatum
[P < 0.01 and P < 0.001, respectively] and the hypothalamus [P < 0.05
and P < 0.001, respectively].
3.3.4. 5HIAA
Measurements of 5HIAA levels revealed a diet effect on the
thalamus and the medulla [F(2,42) = 5.51, P = 0.007; F(2,41) = 3.24,
P = 0.05, respectively]. Methyl donor deficient rats showed lower
5HIAA levels than the others in the thalamus [P < 0.01 and P < 0.05,
respectively] but higher levels than free-feeding rats in the medulla
[P < 0.05].
3.3.5. 5HT
There was a diet effect on 5-HT levels in the medulla
[F(2,41) = 10.87, P = 0.0002] and diet × stress interactions in
the sensorimotor cortex, the striatum, and the cerebellum
[F(2,42) = 3.33, P = 0.04; F(2,42) = 3.24, P = 0.05; F(2,42) = 4.83,
P = 0.01, respectively]. In the medulla, methyl donor deficient rats
had higher 5HT levels than the others [P < 0.01 and P < 0.0001,
respectively]. In the sensorimotor cortex, levels were higher in
methyl donor deficient and free-feeding rats compared to the pair-
fed among non-stressed groups, and the pair-fed rats compared to
free-feeding ones among stressed groups. In the striatum, levels
were higher in the pair-fed rats compared to the free-feeding ones
among non-stressed groups, and, also in the free-feeding rats com-
pared to methyl donor deficient ones among stressed groups. In
the cerebellum, levels were higher for methyl donor deficient and
free-feeding rats compared to pair-fed ones among stressed groups.
3.3.6. 5HIAA/5HT ratio
There were effects of diet conditions on 5HIAA/5HT ratio in
the sensorimotor cortex, the striatum, the hypothalamus, and
the medulla [F(2,42) = 13.11, P < 0.0001; F(2,42) = 4.44, P = 0.02;
F(2,41) = 3.67, P = 0.03; F(2,41) = 6.38, P = 0.004, respectively], as
well as diet × stress interactions in the sensorimotor cortex and
in the medulla [F(2,42) = 8.37, P = 0.0009; F(2,41) = 3.77, P = 0.03,
respectively]. Methyl donor deficient rats showed a lower ratio
than pair-fed and free-feeding rats in the hypothalamus [P < 0.05].
Methyl donor deficient rats had a lower ratio than pair-fed rats
in the sensorimotor cortex and the medulla, among non-stressed
groups [P < 0.001 and P < 0.05, respectively] and also lower than
free-feeding rats, but only in the sensorimotor cortex [P < 0.05].
Among stressed groups, methyl donor deficient rats had a lower
ratio than pair-fed rats in the sensorimotor cortex [P < 0.05], and
also had a lower ratio relative to free-feeding rats in the sensorim-
otor cortex and the medulla [P < 0.001]. Pair-fed rats also showed
a lower ratio than free-feeding rats in the sensorimotor cortex and
medulla [P < 0.01].
4. Discussion
4.1. Plasma homocysteine and corticosterone
The methyl donor deficient diet given over the course of
11 weeks led to a 6-fold increase in plasma homocysteine lev-
els of Wistar rats relative to pair-fed and free-feeding controls
14 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16
independently of exposure to UCMS. The same diet given to mice
for weeks led to a 2-fold increase, also significantly different from
each control group [19]. Since homocysteinemia was observed after
selective folate deficiency from 4 to 8 weeks in rats [29–32], this
may be due to the absence of this vitamin alone, even though it is
likely that this effect was amplified by the additional depletion of
vitamins B2 and B12 as well as choline.
In addition to homocysteine, the methyl donor deficient diet
decreased plasma corticosterone concentrations in non-stressed
pair-fed and free-feeding rats. UCMS-induced higher plasma corti-
costerone levels were similar to those reported by Bielajew et al.
[33] and Grippo et al. [34], who used this stress paradigm in rats
during 3 and 4 weeks, respectively. There seems to be a relationship
between higher serum cortisol concentrations and homocysteine-
mia in human subjects, although it is dependent on several factors
such as age and co-morbidities [35,36]. Nevertheless, despite of the
reduction of serum folate and vitamin B12 levels, subchronic injec-
tions of either adrenocorticotrophic hormone (ACTH) or cortisol
during four days had no impact on plasma homocysteine levels in
human subjects [37], such as UCMS in rats. In contrast, the reduc-
tion of plasma corticosterone levels in rats exposed to the methyl
donor deficient diet may be due to changes in brain monoamine
levels and may have influenced behaviors in open-field, elevated
plus-maze, and forced swim tests.
4.2. Exploratory activity and forced swimming
In the open-field, the methyl donor deficient group reared more
often than either pair-fed or free-feeding groups among stressed
but not among non-stressed rats. This effect was not found on
ambulatory activity and time spent inside the central square. UCMS
interacted with diet on the rearing measure with no effect on
ambulatory activity, unlike the reduction of ambulatory activity
previously found in rats exposed to this paradigm [38]. Conversely,
stress-resistant rats increased novelty-seeking behaviors in the
open-field and elevated plus-maze tests following exposure to
UCMS in previous studies [39]. Diet had no effect on rearing in non-
stressed rats, which is consistent with data found in non-stressed
mice [19]. However, the use of this diet in mice increased open-field
ambulation and enclosed arm entries. Besides species differences
which might explain these results, diet exposure began earlier (1
month versus 3) and lasted longer (8 weeks versus 5) in rats than
Balb/c mice, known for its low levels of activity relative to other
strains such as C57BL/6 [40]. Food restriction alone has long been
shown to induce hyperactivity in rats [41], but this result was not
found in our study, except for an increase in rearing behaviors in
stressed rats exposed to methyl donor deficiency.
There was an increased vertical exploration and open arm explo-
ration in the elevated plus-maze: the methyl donor deficient group
entered more open arms and spent more time in the open arms than
pair-fed or free-feeding rats regardless of UCMS, which is consistent
with decreased anxiety. In studies with C57BL/6 and Balb/c mice,
rearing activity was positively correlated with open and enclosed
arm entries [40]. However, these measures were not correlated
in Wistar rats selectively bred for high rearing activity, who were
more active than those with low rearing activity, but whose behav-
ior did not differ from typical Wistar rats in open arm exploration
[42]. Inoue et al. [43] reported that acute and subchronic (10 days)
food restriction in rats increased time spent in the open arms, but,
in the present conditions, this result was observed only in the defi-
cient groups. The decreased anxiety rates may be due to reduced
corticosterone levels found in these groups, because, when the hor-
mone was injected in the amygdala of rats, open arm exploration
decreased [44].
In addition to exploratory activity, the vitamin B-deficient diet
influenced the forced swim test, which reflects depression-like
activity. In particular, the UCMS-exposed methyl donor deficient
group spent less time swimming, more time climbing, and climbed
more often than the two control groups, but this result was
not found among non-UCMS-exposed rats. These results are very
similar to those found in the open-field, in which only the UCMS-
exposed methyl donor deficient group reared more often. Alcaro
et al. [45] observed increased immobility time in C57BL/6 mice
during forced swimming after 12 days of food restriction, and this
effect was observed in our pair-fed or deficient groups. Although
our UCMS-exposed methyl donor deficient group spent less time
swimming, it did not spend more time immobile, but rather more
time climbing, which seemed like escape attempts.
In the forced swimming test, increased climbing time is
a characteristic of antidepressant medication. Excessive and
persistent climbing may be due to low corticosterone levels
found in methyl donor deficient rats. Thus, lower activation
of the hypothalamic–pituitary–adrenal axis observed in methyl
donor deficiency rats may have induced an adaptative and
antidepressant-like behavior. However, hyperhomocysteinemia
could also explain this overactivity in the forced swimming
test through a neurotoxic effect. Indeed, homocysteinic acid, an
oxidized derivative of homocysteine, acts as an excitatory neuro-
transmitter and is known to induce flight reactions in rats when
injected into the dorsal periaqueductal gray area [46]. Increased
climbing in our hyperhomocysteinemic rats may have been caused
by glutamatergic overactivity or altered monoamine transmission.
4.3. Biogenic amines and metabolites in brain
Diet-induced alterations in biogenic amine concentrations may
be responsible for different plasma corticosterone levels and behav-
iors. Relative to pair-fed and free-feeding rats and independently
of UCMS, methyl donor deficient rats had lower DOPAC levels and a
lower DOPAC/dopamine ratio in the hypothalamus, the ratio being
also lower in the striatum. Although decreased dopamine turnover
estimated by its major metabolite DOPAC in the hypothalamus has
been noted during restricted food intake and 30% of body loss [47],
this result was found only in our deficient group. A decrease in the
DOPAC/dopamine ratio in the hypothalamus may be responsible
for reduced plasma corticosterone concentrations, since dopamine
exerts a positive control on ACTH release [48]. Folate deficiency
and homocysteinemia are liable to disrupt monoamine synthesis
[4,5]. Conversely, folic acid administration is sometimes of benefit
on human hyperactivity symptoms, seemingly characterized by a
hypodopaminergic state [49]. The offspring of dams given a methyl
donor deficient diet accumulated homocysteine in striatal neu-
rons and astrocytes [22]. Homocysteinemia causes neuroexcitatory
effects, notably in the striatum, mediated by N-methyl-d-aspartate
(NMDA) or metabotropic glutamate receptors [50], leading to
neurotoxicity [51,52]. Therefore, changes in dopaminergic trans-
mission in striatum may be responsible for increased rearing
in open-field and increased climbing during forced swimming.
Indeed, peripherally administered dopamine-stimulating drugs
can increase rearing in the open-field and climbing in the forced
swim test [53]. The diet effect superseded any stress effect on
dopaminergic striatal activity, although UCMS may also decrease
this activity [54].
Independently of UCMS and in parallel to dopamine-related
mechanisms, methyl donor deficient rats had lower 5HIAA levels
and a lower 5HIAA/5HT ratio in the thalamus compared to either
control group. The ratio was also lower in the sensorimor cortex, but
only in the subgroup exposed to UCMS. Moreover, the methyl donor
deficient group also increased 5HT levels in the medulla. Since
Ashcroft and Sharman’s pioneering work [55], decreased levels of
5HIAA in CSF of depressive patients has been well established and
could reflect a general decrease in 5HT turnover, but this result was
 H. Javelot et al. / Behavioural Brain Research 261 (2014) 8–16
found only in our deficient group and only in the thalamus and the
sensorimotor cortex. Subnormal levels in the methyl donor defi-
cient group may be due to folate or vitamin B12 deficiency. Indeed,
either folate or vitamin B12 deficiency diminished CSF levels of
5HIAA in humans [56,57] and folate deficiency diminished whole-
brain 5HT levels in rats [56]. This decrease is likely responsible for
depressive symptoms often observed in folate-deprived patients
[4,5]. In addition to the possible influence of B vitamins, 5HIAA-
related changes may have been caused by low corticosterone levels,
as adrenalectomy causes regionally selective changes in 5HT lev-
els or turnover that are reversed following administration of the
hormone [58].
Although it also interacts with diet, the impact of UCMS on
monoamine levels was minimal. Nevertheless, stressed rats had
higher DOPAC levels in medulla, but did not differ from non-
stressed rats regarding 5HT or 5HIAA. Medial and dorsal raphe
neurons projecting to substantia nigra, ventral tegmentum, and
neocortex are usually activated during stressful episodes [59], and
sometimes 5HT turnover increases during chronic stress [60–62]. In
contrast, UCMS has been described as lowering 5HT and 5HIAA lev-
els, or the 5HIAA/5-HT ratio, particularly in the frontal cortex, more
variably in the striatum, the hippocampus or the hypothalamus
[54,63,64]. However, this result was not found here.
In sum, these data indicate that MDD stress-related behaviors
are potentially similar to those reported in agitated depression
with psychomotor agitation and anxiety [65]. These symptoms are
observed in melancholic depression with weight loss and reduced
folate levels [66,67], in major depressive disorders with burst of
anger and hyperhomocysteinemia [21] or in geriatric depression,
highly associated with hyperhomocyteinemia, whose agitated-
form include irritability, pacing and unstoppable worries [68–70].
5. Conclusions
Psychiatric disorders are often due to multiple factors result-
ing from behavioral and neurobiological disturbances. Deficiencies
in B-vitamins and chronic stress could be causative agents act-
ing together in depression and other mood-related disorders.
Our results show that, on one hand, chronic stress exacerbated
the effects of methyl donor deficiency in open-field and forced
swim tests. On the other hand, methyl donor deficiency alone
decreased plasma corticosterone as well as DOPAC and 5HIAA levels
and caused disinhibition in the elevated plus-maze. Our exper-
imental data may therefore have relevance to neuropsychiatric
disorders frequently associated with folate deficiency, hyperhomo-
cysteinemia and psychomotor agitation, such as geriatric agitated
depression.
Acknowledgments
We thank Sophie Hidalgo and Valérie Demade from ETAP
Laboratory – Vandoeuvre-Lès-Nancy for technical assistance in
behavioral testing. We are grateful to Elise Jeannesson and Philippe
Gérard (LNGERE, INSERM U954 – Vandoeuvre-Lès-Nancy) for help
in determining homocysteine and corticosterone concentrations in
plasma samples.
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